RESUMO
Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.
Assuntos
Antígeno B7-1 , Folículo Piloso , Inflamação , Pele , Células-Tronco , Linfócitos T Reguladores , Cicatrização , Animais , Linfócitos T Reguladores/imunologia , Camundongos , Cicatrização/imunologia , Pele/imunologia , Pele/lesões , Pele/patologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Inflamação/imunologia , Folículo Piloso/imunologia , Antígeno B7-1/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reepitelização/imunologia , Movimento Celular/imunologia , Proliferação de CélulasRESUMO
SIGNIFICANCE: Comprehensive single-cell proteomics analyses of lung adenocarcinoma progression reveal the role of tumor-associated macrophages in resistance to PD-1 blockade therapy. See related commentary by Lee et al., p. 2515.
Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Pulmonares , Adenocarcinoma/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Macrófagos , Microambiente TumoralRESUMO
BACKGROUND: Despite the profound number of malignant pleural mesothelioma (MPM) patients now treated with programmed cell death 1 (PD-1) blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remains unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM. METHODS: We generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (4 responders and 4 nonresponders). We used The Cancer Genome Atlas MPM cohort (n = 73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model. RESULTS: Immunogenomic analysis by applying our anti-PD-1 resistance signature to The Cancer Genome Atlas cohort revealed that deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by cyclin dependent kinase 4/6 (CDK4/6) inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a-/- AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth compared with anti-PD-1 or CDK4/6 inhibitor alone. CONCLUSIONS: We identified a therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in more than 40% of patients with MPM who demonstrate loss of CDKN2A.
Assuntos
Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurais , Camundongos , Animais , Quinase 4 Dependente de Ciclina , Nivolumabe , Inibidor p16 de Quinase Dependente de Ciclina/genética , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Mesotelioma/metabolismo , Apoptose , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologiaRESUMO
The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo ß-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer ß-lactamase inhibitors.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Infecções por Enterobacteriaceae/microbiologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/metabolismo , California/epidemiologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Enterobacteriáceas Resistentes a Carbapenêmicos/metabolismo , Criança , Pré-Escolar , Infecções por Enterobacteriaceae/epidemiologia , Feminino , Expressão Gênica , Variação Genética , Genoma Bacteriano/genética , Genótipo , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Fenótipo , Porinas/deficiência , Porinas/genética , Adulto Jovem , beta-Lactamases/deficiência , beta-Lactamases/metabolismoRESUMO
OBJECTIVES: Hyperthermic pleural lavage with povidone-iodine (PVP-I) is utilized to control micrometastatic disease following cytoreductive surgery for thymic epithelial tumours (TETs). Our objective was to investigate whether PVP-I demonstrates direct cytotoxicity against human TET cells. METHODS: Human Met-5A (immortalized mesothelial cell), IU-TAB-1 (thymoma) and Ty-82 (thymic carcinoma) cell lines were treated with serial dilutions of PVP-I (0.01-10%) for 5, 30 and 60 min at 37°C and 42°C. MTT assays and flow cytometry were used to evaluate cell death and apoptosis. Membrane permeability was assayed by intracellular staining of cleaved poly-ADP-ribose polymerase. Cellular fixation was evaluated by membrane disruption of dead cells by dimethylsulphoxide and by comparing cleaved poly-ADP-ribose polymerase staining following PVP-I with known fixatives. RESULTS: MTT assays demonstrated that PVP-I concentrations greater than 0.5% led to rapid cell death in both TET cell lines regardless of temperature. IC50 values following 5 min of exposure to PVP-I were 8.4 mM (0.3%) and 13.3 mM (0.48%) for IU-TAB-1 and Ty-82, respectively and 8.9 mM (0.32%) for MeT-5A. Flow cytometry demonstrated that 5-min exposure of either cell line to 1% PVP-I resulted in profound cell death: 74% and 58% at 5 min and 97% and 95% at 30 min, for IU-TAB-1 and Ty-82 cells, respectively. Resistance of PVP-I-treated cells to dimethylsulphoxide lysis and similar cleaved poly-ADP-ribose polymerase expression following PVP-I and known fixatives revealed cellular fixation as the mechanism of death following PVP-I exposure. CONCLUSIONS: PVP-I results in rapid death of human TET cells and normal mesothelial cells through a cellular fixation mechanism and may, therefore, favourably impact the control of micrometastatic disease following resection of TETs with pleural dissemination.
Assuntos
Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Povidona-Iodo/farmacologia , Neoplasias do Timo/tratamento farmacológico , Anti-Infecciosos Locais/farmacologia , Apoptose , Linhagem Celular Tumoral , Humanos , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias do Timo/patologiaAssuntos
Receptor de Morte Celular Programada 1/antagonistas & inibidores , Timoma/tratamento farmacológico , Timoma/genética , Neoplasias do Timo/tratamento farmacológico , Neoplasias do Timo/genética , Anticorpos Monoclonais Humanizados/administração & dosagem , Humanos , Mutação , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Timoma/imunologia , Neoplasias do Timo/imunologia , Neoplasias do Timo/patologiaRESUMO
Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The noninvasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.
Assuntos
Doenças Transmissíveis/diagnóstico , Biópsia Líquida , Adulto , Idoso , Ácidos Nucleicos Livres , Doenças Transmissíveis/sangue , Doenças Transmissíveis/microbiologia , DNA Fúngico/sangue , DNA Fúngico/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infecções Fúngicas Invasivas/sangue , Infecções Fúngicas Invasivas/diagnóstico , Infecções Fúngicas Invasivas/microbiologia , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Adulto JovemRESUMO
There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (CT ) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy of CT was measured at different CT cutoffs. Using RMEIA as the reference method, a CT cutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improved CT specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve CT toxin specificity. The median CT values were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.
Assuntos
Proteínas de Bactérias/análise , Toxinas Bacterianas/análise , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/diagnóstico , Enterotoxinas/análise , Fezes/química , Adulto , Clostridioides difficile/genética , Enterocolite Pseudomembranosa/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/microbiologia , Feminino , Humanos , Técnicas Imunoenzimáticas/métodos , Masculino , Reação em Cadeia da Polimerase/métodosRESUMO
Health care-onset health care facility-associated Clostridium difficile infection (HO-CDI) is overdiagnosed for several reasons, including the high prevalence of C. difficile colonization and the inability of hospitals to limit testing to patients with clinically significant diarrhea. We conducted a quasiexperimental study from 22 June 2015 to 30 June 2016 on consecutive inpatients with C. difficile test orders at an academic hospital. Real-time electronic patient data tracking was used by the laboratory to enforce testing criteria (defined as the presence of diarrhea [≥3 unformed stools in 24 h] and absence of laxative intake in the prior 48 h). Outcome measures included C. difficile test utilization, HO-CDI incidence, oral vancomycin utilization, and clinical complications. During the intervention, 7.1% (164) and 9.1% (211) of 2,321 C. difficile test orders were canceled due to absence of diarrhea and receipt of laxative therapy, respectively. C. difficile test utilization decreased upon implementation from an average of 208.8 tests to 143.0 tests per 10,000 patient-days (P < 0.001). HO-CDI incidence rate decreased from an average of 13.0 cases to 9.7 cases per 10,000 patient-days (P = 0.008). Oral vancomycin days of therapy decreased from an average of 13.8 days to 9.4 days per 1,000 patient-days (P = 0.009). Clinical complication rates were not significantly different in patients with 375 canceled orders compared with 869 episodes with diarrhea but negative C. difficile results. Real-time electronic clinical data tracking is an effective tool for verification of C. difficile clinical testing criteria and safe reduction of inflated HO-CDI rates.
Assuntos
Infecção Hospitalar/epidemiologia , Diarreia/diagnóstico , Diarreia/epidemiologia , Processamento Eletrônico de Dados/métodos , Enterocolite Pseudomembranosa/diagnóstico , Enterocolite Pseudomembranosa/epidemiologia , Medicina Baseada em Evidências/métodos , Sistemas de Informação Hospitalar , Idoso , Antibacterianos/uso terapêutico , Clostridioides difficile/efeitos dos fármacos , Diarreia/tratamento farmacológico , Enterocolite Pseudomembranosa/tratamento farmacológico , Feminino , Humanos , Laxantes/uso terapêutico , Masculino , Metronidazol/uso terapêutico , Pessoa de Meia-Idade , Vancomicina/uso terapêuticoRESUMO
BACKGROUND: The Clostridium difficile rate in symptomatic patients represents both those with C. difficile infection (CDI) and those with colonization. To predict the extent of CDI overdiagnosis, we compared the asymptomatic colonization rate to the symptomatic positivity rate in hospitalized patients using nucleic acid testing. METHODS: Between July 2014 and April 2015, formed stool samples were collected from asymptomatic patients after admission to 3 hospital wards at the Stanford Hospital. Stool samples from symptomatic patients with suspected CDI in the same wards were collected for testing per provider order. The GeneXpert C. difficile tcdB polymerase chain reaction (PCR) assay (Cepheid, Sunnyvale, CA, USA) was performed on all stool samples and PCR cycle threshold was used as a measure of genomic equivalents. Chart review was performed to obtain clinical history and medication exposure. RESULTS: We found an asymptomatic C. difficile carriage rate of 11.8% (43/365) (95% confidence interval [CI], 8.5-15.1%) and a positivity rate in symptomatic patients of 15.4% (54/351) (95% CI, 11.6-19.2%; P=0.19). The median PCR cycle thresholds was not significantly different between asymptomatic carriers and symptomatic positives (29.5 versus 27.3; P=0.07). Among asymptomatic patients, 11.6% (5/43) of carriers and 8.4% (27/322; P=0.56) of noncarriers subsequently became symptomatic CDI suspects within the same hospitalization. Single and multivariate analysis did not identify any demographic or clinical factors as being significantly associated with C. difficile carriage. CONCLUSIONS: Asymptomatic C. difficile carriage rate was similar to symptomatic positivity rate. This suggests the majority of PCR-positive results in symptomatic patients are likely due to C. difficile colonization. Disease-specific biomarkers are needed to accurately diagnose patients with C. difficile disease.
Assuntos
Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/microbiologia , Infecção Hospitalar/microbiologia , Ácidos Nucleicos/genética , Idoso , Infecções Assintomáticas , Portador Sadio/microbiologia , Enterocolite Pseudomembranosa/microbiologia , Feminino , Hospitalização , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Fatores de RiscoRESUMO
Understanding the contribution of relapse and reinfection to recurrent Clostridium difficile infection (CDI) has implications for therapy and infection prevention, respectively. We used whole genome sequencing to determine the relation of C. difficile strains isolated from patients with recurrent CDI at an academic medical center in the United States. Thirty-five toxigenic C. difficile isolates from 16 patients with 19 recurrent CDI episodes with median time of 53.5days (range, 13-362) between episodes were whole genome sequenced on the Illumina MiSeq platform. In 84% (16) of recurrences, the cause of recurrence was relapse with prior strain of C. difficile. In 16% (3) of recurrent episodes, reinfection with a new strain of C. difficile was the cause. In conclusion, the majority of CDI recurrences at our institution were due to infection with the same strain rather than infection with a new strain.